Our research program addresses basic molecular and physiological processes of nociceptive transmission in the central nervous system and new, effective ways to treat intractable pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons that innervate the skin and deep tissues and their connections in the dorsal spinal cord, which is the first site of synaptic information processing for pain. Our research has identified it as a locus of neuronal plasticity and altered gene expression in persistent pain states. The regulation of transduction of physical pain stimuli is also under investigation using cloned thermal and chemo-responsive ion channels ectopically expressed in heterologous cell systems and naturally expressed in primary cultures of dorsal root ganglion. Our goals are (1) to understand the molecular and cell biological mechanisms of acute and chronic pain at these two basic levels of the nervous system and (2) to use this knowledge to devise new treatments for pain. We address the latter goal in a translational research and human clinical trials program designed to evaluate new analgesic treatment for severe pain. The treatment we are developing is based on our studies of the molecular mechanisms of pain transduction through the vanilloid receptor 1 (TRPV1). This molecule is a heat-sensing calcium/sodium ion channel that converts painful heat into nerve action potentials by opening the channel and depolarizing pain-sensing nerve terminals. Channel opening is also stimulated by capsaicin which is a vanilloid chemical and the active ingredient in hot pepper. We use a very potent vanilloid analog to prop open the channel causing death of a specific class of pain-sensing neurons, yet allowing mechanical and high temperature heat pain sensations and other somatosensory and proprioceptive sensations to remain intact. We have established an inter-institute working group with NIDA's Division of Pharmaco-Therapeutics and Medical Consequences of Drug Abuse to bring the treatment to human clinical trial. The working group consists of experts on medical, neurobiological, toxicological, chemical and formulation issues as well as anesthesiologists, pharmacologists and pathologists from our group. Over the course of this year, we also established mechanisms for obtaining the natural product from which the active drug is extracted and procedures for isolation, purification and formulation of the drug product such that it will be compliant with Food and Drug Administration (FDA) regulations. We are presently finalizing the toxicology study, the Investigational New Drug Application with the FDA and the Human Clinical Protocol with the NCI's IRB. The treatment we have devised may be a very effective approach to control many types of chronic pain especially those associated with cancer, arthritis, tempromandibular joint disorders, trigeminal neuralgia and chronic neuropathic pain problems. Underlying the translational studies are the questions of molecular regulation in chronic pain and mechanisms of pain transduction in peripheral nerve ending. These questions are addressed using subtraction cloning, differential hybridization and gene arrays, and neurophysiological measurements such as calcium imaging in live cells. The physiological stdies have focused on he multiple intracellular pools of calcium that can be activated by vanilloid agonists and the interaction of these pools with the plasma membrane localized TRPV1 and TRPV1 located on the endoplasmic reticulum. Activation of TRPV1 in both locations is a factor that underlies the efficacy of TRPV1 agonists at inducing calcium cytotoxicity in the above translational studies. The molecular studies reveal a more dynamic modulation of gene expression in dorsal root ganglion than previously hypothesized, for example, in a matter of hoours we observe up-regulation of the receptor for Neuropeptide FF, which is known to be involved in opioid modulation of pain. In addition to pain, these studies fundamentally explore the molecular basis of synaptic plasticity. New roles for the calcium and arachidonic acid binding proteins S100A8 and S100A9 in spinal cord and dorsal root ganglion have also been discovered. We hypothesize modularity in the neuronal response new levels of synaptic or pharmacological input (e.g. learning, neurological disorders, drug abuse). The "generic" alterations are combined with modulation of tissue-specific genes to meet the demands generated by the new level of stimulation. This will lead to a deeper understanding of molecular mechanisms that trigger and sustain chronic pain.